Over the last years, several epidemic studies have been correlating the Mediterranean diet with the low frequency of heart diseases, atherosclerosis and defined types of cancer. A particular characteristic of the Mediterranean diet is the use of olive oil, directly consumed or used for cooking (Visioli et al., 2002; Owen et al., 2000).
Studies concerning biophenolic compounds present in olives and olive leafs, draw researchers to recognize their biological properties, which were also associated with the positive health properties of olive oil. Therefore, the bioactive compounds present in olives and olive oil were recognized as important targets for the pharmaceutical and the food industry (Schieber et al., 2001). However, 98% of these biophenolic compounds are lost during olive oil production and remain in vegetation water and/or solid residues resulting from olive processing (Rodis et al., 2002).
The compounds present in higher concentrations in the solid residues are glycosylated secoiridoids, while in vegetation water the compounds with higher concentration are secoiridoids derivatives, mostly hydroxytyrosol and oleoeuropein (Mullinacci et al., 2001).
The properties of the solid residues obtained during olive oil production, as well as extracts obtained from them, have been studied (Visioli et al., 1999); in particular, their anti-bacterial activity has been shown (Ramos-Cormenzana et al., 1996), and associated with the presence of oleoeuropein and hydroxytyrosol; this last compound was referred to be the compound with a higher bio-activity (Bisignano et al., 1999).
Hydroxytyrosol has been also referred as a potent chemo-preventive agent (Manna et al., 2000), and considered as the component present in olive oil residues with higher anti-oxidant potency. The first recognized properties of hydroxytyrosol were its ability to prevent the oxidation of the low density lipoprotein (LDL) (Visioli and Galli, 1998) and the aggregation of blood platelet (Petroni et al., 1995). Mana et al. (2000) proved that this compound is able to protect several cellular human systems from the toxicity induced by reactive oxygen species. The ability of hydroxytyrosol to induce DNA modifications has been also investigated (Aruoma et al., 1999 and Deima et al., 1999).
Visioli et al. (2000) has also shown that, depending on the dosage, this biophenolic compound is well absorbed by humans, being excreted in urine as glucuronate conjugates.
Nowadays, olive oil is exclusively produced by using mechanical and physical methods, that consist on pressing the fruit (pulp and stones) until obtaining a homogeneous slurry, which is then processed for phase separation.
The traditional phase separation step uses hydraulic presses, which have been replaced by continuous centrifugation. The continuous process is also named three-phase process, when olive oil, vegetation water and olive cake are obtained as final products, or as two-phase process when the final product streams are olive oil and olive cake.
In the three-phase system, it is added water to the olive slurry and this mixture is then processed by a horizontal centrifuge, where the solid phase is separated from the oily must. This must is then processed by a vertical centrifuge, where the olive oil is separated from the vegetation water.
The most common method, nowadays, is the two-phase method because it involves a lower consumption of water. Consequently it produces a lower amount of residual water. This process uses two-phase centrifuges that separate olive oil and olive cake. The olive cake is a semi-solid residue with a slurry-type aspect.
Several patents have been published, which present methods for the recovery of fenolic compounds from olive tree residues. The U.S. Pat. No. 6,361,803 describes a method for the recovery of antioxidant compounds from olive residues. This method consists on a preliminary extraction with an aqueous solvent, being the extract produced fed to an adsorption column, in order to retain the compounds of interest. These compounds are recovered subsequently by eluting an organic solvent through the adsorption column. The U.S. Pat. No. 6,849,770 describes a method for the recovery of hydroxytyrosol by a chromatographic method using methanol or water/ethanol mixtures as elution solvents.
The WO0218310 describes a method of obtaining a hydroxytyrosol-rich composition from vegetation water using a previously patented method (U.S. Pat. No. 5,490,884), known as Porocrit. Extraction of the target compounds from vegetation water is achieved using supercritical fluids, such as carbon dioxide, and porous membranes instead of contacting columns. Instead of dispersing the phases, the liquid is fed continuously through porous polypropylene membranes configured as hollow fibre bundles or spiral wound sheets. The liquid passes through the porous membranes within a pressurized module, while supercritical carbon dioxide flows counter-currently on the other side of the membrane. It is important to notice, according to the authors of this patent, that the pressure in the module is essentially the same, so that the extraction is driven by the concentration gradient between the fluid and the supercritical carbon dioxide and not by a pressure gradient between the two sides of the membrane. The extract may be recovered by vaporizing the carbon dioxide for recycling. Additionally, the U.S. Pat. No. 5,714,150 describes a method for extraction of oleuropein from leafs of the olive tree by using water/ethanol mixtures.
The use of membrane filtration methods and their integration with other techniques, such as centrifugation, have been reported by research groups aiming to develop processes for treatment of olive mill wastewaters.
The WO0218310 disclosed process differs from the one of the present invention mainly because it does not include a reverse osmosis operation being this operation comprised in the second step of the present invention. Other differential aspect of this process is that the pressure in the two opposite sides of the membrane is essentially the same, so that the extraction is driven by the concentration gradient between the fluid and the supercritical carbon dioxide and not by a pressure gradient between the two sides of the membrane. Consequently, the product obtained by the process described in of WO0218310 is substantially different from the extract obtained according to the process of the present invention, which, most preferentially, excludes compounds with a molecular weight above 300 Da.
Drouiche et al. (2004) describe the use of ultrafiltration to remove the particles and organic compounds from vegetation water. The work published by Turano et al. (2002) describes a process which integrates centrifugation and ultrafiltration for treatment of vegetation water. In this integrated process, centrifugation allows to remove the suspended solids fraction protecting the ultrafiltration membrane from severe fouling problems caused by these particulates.
DellaGreca et al. (2001 and 2004) report an analytical method developed for characterization of vegetation water. This method involves the fractionation of vegetation water in order to obtain isolated, individual components, present in the water. A series of membrane techniques is used for this purpose.
Document US 2003/0108651 refers to a process different from the one described in the present invention, namely because it does not include a reverse osmosis operation, which is comprised in the second step of the present invention. As a consequence of using different processes, the complex extract obtained according to the process of the present invention is different from the product obtained by the process described in US 2003/0108651, which describes that the method comprises fractionating the incubated vegetation water to separate hydroxytyrosol from other components. Additionally, this method uses for fractionation and separation of hydroxytyrosol the incubation of vegetation water with an organic solvent, namely ethyl acetate, and further comprises the purification of hydroxytyrosol by chromatography. Contrarily, the present invention does not comprise the separation of hydroxytyrosol nor its purification by chromatography or by any other means once it aims to obtain a biological extract, which comprises hydroxytyrosol and other bioactive compounds.
EP 1 623 960 A1 describes a process for the treatment of oil mill wastewaters allowing for the recovery of highly pure tyrosol and hydroxytyrosol. In order to obtain such product, this process uses membrane techniques. Additionally, the compound tyrosol recovered from oil mill wastewaters is catalytically converted to hydroxytyrosol. Therefore, this process aims for obtaining highly pure compounds instead of natural complex extracts from olive oil sub-products.
Skaltsounis, L. et al. (MINOS PROJECT Process development for an integrated olive mill waste management recovering natural antioxidants and producing organic fertilizer) describes an adsorption process, since it uses a resin to capture polyphenols from olive mill wastewaters. The polyphenols captured in the resin medium are therefore recovered by using an organic solvent. Further, these polyphenols are thermally recovered from the organic solvent used and separated by chromatography. Nanofiltration/reverse osmosis are referred in this project as a way to treat the resin brine outflow effluent, which is rich in mineral salts (counter-ions from the resin material) but does not contain polyphenols. Thus, the above mentioned process is totally different from the one described in the present invention due to the use of that resin to isolate the olive mill wastewaters polyphenols instead of using a two-step process consisting in extracting the biological compounds by means of a supercritical fluid extraction column or a nanofiltration unit, and then using a reverse osmosis unit to obtain a biological extract containing hydroxytyrosol.
A process using clean technologies able to recover, from olive oil residues, a complex, natural extract, rich in hydroxytyrosol and containing other bioactive compounds with desirable properties, while assuring the rejection of compounds with higher molecular weight and detrimental biological properties is not available.
Hydroxytyrosol rich concentrates are available in the market as a dietetic supplement. One of these products is produced according with the patented process WO 0218310 previously referred. It is claimed that this concentrate can be used as a natural anti-bacterial, anti-viral and/or anti-fungi agent in agriculture, as well as a therapeutical agent or food additive.
In fact, studies were carried-out in order to compare the result of the process disclosed in document WO0218310, a commercial product designated as Hydrox®, Creagi, and the extract obtained by the process of the present invention. Both products were tested for the cytotoxicity on CACO-2 (human colon) cell line and anti-proliferative activity on HT-29 (human colon cancer cell line. Results show that both extracts are not cytotoxic for the CACO-2 (human colon) cell line. However, the proliferative inhibitory effect of the natural extract produced by the present invention, on the tested human cancer cell line, is higher (110% higher for concentrations of polyphenols above 200 mg/L) than the biological activity exhibited by the product obtained according to the process described in WO0218310.
Furthermore the present invention uses as feedstock any type of olive tree residue including solid residues and does not aim at the isolation of hydroxytyrosol by itself, but at obtaining a complex extract rich in this compound comprising also other bioactive compounds.